Despite their low natural abundance, rare sugars hold enormous potential for practical application. Some of the use of the rare sugars ranges from low caloric sweetener to medical application.
Research on rare sugars is progressing rapidly and the application of these rare sugars has been spread quite widely to sweeteners, functional foods, medicines, cosmetics, and surprisingly to agrochemical fields. In addition, rare sugars can be used as starting materials for the synthesis of intriguing natural products with important biological activities. Unfortunately, most rare sugars are quite expensive, and their synthetic routes are both limited and costly due to the expense of costly starting materials.
D-Psicose is one of the important hexose rare sugar useful as low caloric sweetener, anti-oxidant, and as an agrochemical. Psicose, a carbon-3 epimer of Fructose, is a rare monosaccharide. In nature, the Psicose is present as a non-fermentable constituent of cane molasses in a very minute quantity, a sugar moiety of the nucleoside antibiotic psicofuranine, and as free sugar in wheat and itea plants. Psicose has the unique property of being an ideal sucrose substitute as a non-caloric sweetener for weight reduction and a nontoxic sugar.
Compared with sucrose, it has 70% the sweetness but provides no energy due to its suppressive effect toward hepatic lipogenic enzymes. Furthermore, it has been observed that foods supplemented with Psicose exhibit higher antioxidant activity.
Moreover, Psicose can be used as a precursor in the synthesis of xylosylpsicoses, which are promising candidates for prebiotics, cosmetics and therapeutic uses. Allose is another important hexose rare sugars useful as low caloric sweetener. Allose, an aldohexose, is C-3 epimer of D-glucose, exists rarely in nature but has been isolated from the leaves of the African shrub Protea rubropilosa. Allose has the unique property of being an ideal sugar substitute as a non-caloric sweetener for weight reduction and a nontoxic sugar. Moreover Allose has beneficial activities, including anti-cancer, anti-tumour, anti-inflammatory, anti-oxidative, anti-hypertensive, cryoprotective, and immunosuppressant activities. Allose is as sweet compared to sucrose but provides no energy due to its suppressive effect toward hepatic lipogenic enzymes.
The enzyme responsible for bioconversion of ketose to its corresponding epimeric ketose (fructose to psicose) form have been reported from different microorganisms such as Agrobacterium tumefaciens, Rhodobacter Sphaeroides, Ruminococcus sp, RHIzobium leguminosarum, Clostridium cellulolyticum H10 and Pseudomonas cichorii ST-24. U.S. Pat. No. 5,679,562 discloses enzyme from Pseudomonas cichorii ST-24 having ability to convert ketose sugars to their corresponding epimeric form. U.S. Pat. No. 5,811,271 described the conversion of L-ketohexoses to its epimeric form with the D-ketohexose 3-epimerase and reported the affinity of the enzyme towards tagatose. The same enzyme is sometimes referred as D-tagatose-3-epimerase due its more specificity towards D-tagatose compared to Fructose. Similarly the enzymes responsible for bioconversion of ketose to aldose (Psicose in to Allose) form have been reported from different microorganisms as well such as Escherichia coli, Salmonella, Pseudomonas spp and Thermoanaerobacterium saccharolyticum. EP 0807682 discloses the ribose isomerase from Acinetobacter calcoaceticus LR7C capable of converting L-ribose into L-ribulose and vice versa. EP 1589102 disclosed DNA sequence of L-rhamnose isomerase derived from Pseudomonas stutzerii. 
The mass production of pure Psicose and Allose is critical to meet the commercial value due to insufficient production of enzyme as biocatalysts. Therefore heterologous expression of such enzymes is extremely desired to design a cost effective and much safe bioconversion process. Heterologous expression of gene products in different expression system is sometimes limited by the presence of codons that are infrequently used in other organisms. Expression of such genes can be enhanced by systematic substitution of the endogenous codons with codons over represented in highly expressed prokaryotic genes. Redesigning a naturally occurring gene sequence by choosing different codons without necessarily altering the encoded amino acid sequence often dramatically increased protein expression levels. One disadvantage in biocatalyst used in production of low caloric sugar such as Psicose are the production cost of the enzyme due to low expression level of enzymes in native or heterologous organisms. In addition, due to the fact that the inter conversion between Fructose and Psicose is an equilibrium process, the large scale and high yield production of Psicose remains quite challenging.
Even though the enzymes are known that are capable of catalyzing the rare sugars but the gap still remain in mass production of enzymes and difficulties in their expression level besides the problems relating to the fact that the inter conversion between Fructose and Psicose is an equilibrium process.
It is understood that most of these enzymes do not get expressed at industrial scale to be used as a biocatalyst for bioconversion of sugars.
The inventors has identified the production constrain of ketohexose sugar which is a bottleneck for industrial scaling up and identified the expression level in heterologous is low for certain nucleotide which are less preferred. In order to overcome such problem, the nucleotide sequence obtained from Pseudomonas cichorii ST-24 which encodes for the enzymes responsible for bio-conversion were modified to increase the expression level substantially. Such modification resulted in better expression of the enzymes D-tagatose 3-epimerase of Pseudomonas cichorii and rhamnose isomerase of Pseudomonas stutzeri in E. coli. The E. coli host organism used in the invention is JM109 (a K-12 E. coli strain) was used for heterologous expression of recombinant D-tagatose 3-epimerase and rhamnose isomerase. It has been shown the E. coli K-12 cannot be converted into an epidemic pathogen by laboratory manipulation with r-DNA molecules and it will not colonize the human intestinal tract.
The present invention offers an alternative process for producing rare monosaccharides, in which the enzymes were expressed in E. coli at a higher level by modifying the gene sequence. In other words, the present research has made and effort to genetically modify the gene responsible for the production of enzymes, namely d-tagatose 3-epimerase to be used in bioconversion of fructose to psicose. The genetic modification has resulted in increase in expression of protein in E. coli host.